This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The skin squamous cell carcinoma (SSCC), which is derived from malignant keratinocytes, is a major type of skin cancer, more than 1 million cases of which are diagnosed annually within the US. The long-term goals of this project are to define the role of the human alkaline ceramidase 1 (ACER1) in suppressing SSCC, and to develop this concept into novel approaches for the prevention and treatment of SSCC. ACER1 is our newly cloned ceramidase that catalyzes the hydrolysis of ceramide to generate free fatty acid and sphingosine (SPH). Our published data demonstrate that ACER1 is a skin-specific ceramidase that plays an important role in the growth arrest and differentiation of epidermal keratinocytes. In situ hybridization revealed that ACER1 mRNA is expressed at much higher levels in the differentiating and differentiated cell layers of the epidermis than the proliferating basal cell layer. ACER1 expression in cultured keratinocytes is markedly upregulated by extracellular Ca2+, a key physiological inducer of the growth arrest and differentiation of keratinocytes. This upregulation is inhibited by epidermal growth factor (EGF), which promotes keratinocyte proliferation but suppresses differentiation. ACER1 knockdown by RNA interference increases keratinocyte proliferation and suppresses keratinocyte differentiation in response to extracellular Ca2+ elevation. Our preliminary data suggest that ACER1 downregulation may contribute to both hyperproliferation and dedifferentiation of SSCC cells. ACER1 expression is suppressed in all SSCC cell lines that we examined. The expression of the mouse alkaline ceramidase 1 (Acer1) is also substantially down-regulated in chemically induced skin papillomas. Restored expression of ACER1 in SSCC cells not only inhibit the proliferation of SSCC cells but also induces the expression of differentiation markers, such as keratin 1. Our hypothesis is that ACER1 acts as a "tumor suppressor" to inhibit SSCC development and progression through its ability to induce the growth arrest and differentiation of keratinocytes. We will test this hypothesis with the following Specific Aims. Specific Aim 1 Establish that ACER1 downregulation leads to epidermal hyperplasia We will determine whether ACER1 knockdown by RNA interference in keratinocytes causes epidermal hyperplasia in regenerated human skin grafts on a severe combined immunodeficiency (SCID) mouse. Specific Aim 2 Establish that ACER1 plays a tumor suppressor role in SSCC development We will use ACER1 transgenic mice that express the human transgene ACER1 specifically in the epidermis and investigate whether the chemically induced formation of skin tumors is inhibited or reduced in ACER1 transgenic mice. The proposed research in this application is of great importance as the results generated from the experiments will provide an important molecular foundation for the prevention and treatment of SSCC.